X-ray diffraction from permeabilized skeletal muscle fibers associated with each of the eight intermediate states of the ATP hydrolysis cycle has been completed by this group during the past few years,. In addition to identifying filament structures with biochemical intermediate states, the group has established that the filament structures are related to the atomic structures of the myosin head. The open/closed conformations of S1 and the disorder/order equilibrium in the myosin filament are closely correlated. To our knowledge, this is the first time that the structure of the myosin filament in relaxed muscle, the atomic structures and the biochemical states of myosin S1 are tied together. There are other factors that could affect the order &#61527; disorder in the myosin filament. Phosphorylation of the myosin regulatory light chain (RLC) in the skeletal and cardiac myosin is correlated with post-tetanic potentiation and the isometric twitch tension. RLC phosphorylation also increases force generation at submaximal level of Ca2+ activation (a left-shift in the force-PCa curve) and reduces the effect of sarcomere length on force. It has been suggested that phosphorylation disorders the myosin filament, moving myosin heads away from the myosin filament, such that the closer proximity improves the probability of interaction with actin. Phosphorylation of the RLC could, therefore, lead to a structural pathway in modulating force levels independent of the structural cycle associated with ATP hydrolysis. Structural studies have been limited to isolated filaments. The key factors for force generation such as the number of cross-bridges attached, the filament structures and the lattice spacing have to be investigated using muscle cells. During FY2007, studies on the effects of phosphorylation on the myosin filament structure were initiated. Protocol for phosphorylation was mostly developed for single muscle cell, while our experiments involve multi-cells. Protocols for phosphorylating RLC in a bundle of muscle fibers and quantifying the level of phosphorylation have been established. X-ray diffraction from permeabilized muscle was obtained to determine the degree of orderliness of the disposition of the myosin heads on the filaments. The X-ray data were correlated with the phosphorylation level of the myosin RLC. The results showed that phosphorylation of the RLC brings about disorder in the distribution of the myosin heads. It also changes the separation distances between the filaments and the fraction of myosin heads bound to actin in a relaxed muscle. The latter result could provide an explanation of increased calcium sensitivity by RLC phosphorylation, particularly in cardiac muscle.